Ion mobility and mass spectrometer

ABSTRACT

An ion mobility and mass spectrometer instrument includes an ion source region coupled to an ion mobility spectrometer having an ion outlet coupled to a quadrupole mass filter. An output of the filter is coupled to a collision cell which has an ion outlet coupled to an ion acceleration region of a mass spectrometer such as a time of flight mass spectrometer. The instrument is particularly well suited for sequencing analysis wherein a sample is ionized and a resulting three-dimensional ion spectrum (ion intensity vs. ion mobility and ion mass) is observed. If the spectrum reveals that no ions overlap in mobility values, the collision cell is filled with a suitable buffer gas and the instrument is reactivated whereby a complete three-dimensional spectrum of parent and daughter ions results. If, however, the original spectrum reveals that two or more ions overlap in ion mobility values, the collision cell is filled with a buffer gas and the quadrupole mass filter is controlled to selectively filter out all but one of the ions having overlapping mobility values. The instrument is reactivated, and the quadrupole mass filter is selectively controlled, as many times as mobility overlap occurs to thereby provide complete three-dimensional spectra of parent and daughter ions resulting from fragmentation. Various configurations of mass filter, ion trap and collision cell positioning, relative to the ion mobility and mass spectrometer instruments, are contemplated.

CROSS-REFERENCE TO RELATED U.S. APPLICATION

This is a continuation-in-part of co-pending U.S. patent applicationSer. No. 08/867,245, filed Jun. 2, 1997, and entitled HYBRID IONMOBILITY AND MASS SPECTROMETER.

FIELD OF THE INVENTION

The present invention relates generally to instrumentation forcharacterization of molecules based on their structures andmass-to-charge ratios as gas-phase ions, and more specifically to suchinstrumentation which provides for rapid and sensitive analysis ofcomposition, sequence, and/or structural information relating to organicmolecules, including biomolecules, and inorganic molecules.

BACKGROUND OF THE INVENTION

Biological molecules, such as DNA, RNA, proteins, carbohydrates andglycoconjugates, are comprised of repeating subunits typically referredto as residues. The sequence of such residues ultimately defines thestructure and function of the biomolecule and determines how it willinteract with other molecules.

A central part of almost all conventional sequencing strategies is theanalysis of complex sets of sequence-related molecular fragments bychromatography or by polyacrylamide gel electrophoresis (PAGE).PAGE-based automated sequencing instruments currently exist andtypically require a number of fluorescent dyes to be incorporated intothe base-specifically terminated biomolecule product, which is thenprocessed through the polyacrylamide gel. The discrete-length productmolecules are detected near the bottom of the gel by their emittedfluorescence following excitation by a radiation source.

Such automated instruments are typically capable of generating sequenceinformation for biomolecules having 500 or more residues at a rate of10-20 times faster than manual methods. However, both the manual andautomated PAGE techniques suffer from several drawbacks. For example,both approaches are labor-intensive since a gel must be prepared foreach sequencing run. Also, while automated PAGE systems may offer fasteranalysis times than a manual approach, the accuracy of such systems islimited by artifacts generated by non-uniform gel matrices and otherfactors. Such automated systems are generally not equipped to accuratelyprocess the effects of such artifacts, which are typically manifested as“smiling” compressions, faint ghost bands, and the like. Manualinterpretation of such results is therefore often required whichsignificantly increases analysis time.

Researchers have, within the past several years, recognized a need formore rapid and sensitive techniques for analyzing the structure andsequences of biomolecules. Mass spectrometry (MS) techniques, such astime-of-flight mass spectrometry (TOFMS) and Fourier Transformion-cyclotron-resonance mass spectroscopy, are well known techniques forquickly and accurately providing ion mass information from whichsequence and structural determinations can be made. As is known in theart, TOFMS systems accelerate ions, via an electric field, toward afield-free flight tube which terminates at an ion detector. Inaccordance with known TOFMS principles, ion flight time is a function ofion mass so that ions having less mass arrive at the detector morequickly than those having greater mass. Ion mass can thus be computedfrom ion flight time through the instrument. FIG. 1 demonstrates thisprinciple for a cytochrome-c sample, having a known mass to charge ratio(m/z) of 12,360 da, and a lysozyme sample, having a known mass to chargeratio (m/z) of 14,306 da. In FIG. 1, signal peak 10, having a flighttime of approximately 40.52 μs corresponds to the lighter cytochrome-csample, and signal peak 12, having a flight time of approximately 41.04μs, corresponds to the heavier lysozyme sample.

Due to the significantly decreased sample preparation and analysis timesof MS techniques over the above-described PAGE technique, several MSsequencing strategies have recently been developed. Such MS sequencingtechniques are generally operable to measure the change in mass of abiomolecule as residues are sequentially removed from its end. Examplesof two such techniques, each involving elaborate pre-MS processingtechniques, are described in U.S. Pat. No. 5,210,412 to Levis et al. andU.S. Pat. No. 5,622,824 to Köster.

In order to provide for the capability of determining sequence andstructural information for large biomolecules, it has been recognizedthat MS techniques must accordingly be capable of generating large ions.Currently, at least two techniques are known for generating large ionsfor spectral analysis; namely electrospray ionization (ESI) and matrixassisted laser desorption ionization (MALDI). While both large iongenerating techniques are readily available, known MS techniques arelimited in both the quantity and quality of discernable information.Specifically, for large biomolecules, defined here as those containingat least 50 residues, mass spectra of parent and sequence relatedfragment ions become congested to the degree that mass (TOF) peaksoverlap.

One solution to the problem of congested mass spectra is to increase themass resolution capability of the MS instrument. Recent efforts atincreasing such resolution have been successful, and complete sequenceinformation for a 50 base pair DNA has been obtained using a FourierTransform ion cyclotron resonance (FTICR) instrument. However, suchinstruments are extremely expensive, not readily available, and becauseof their extremely high vacuum requirements, they are generally notsuitable for routinely sequencing large numbers of samples.

Another solution to the problem of congested mass spectra is topre-separate the bulk of ions in time prior to supplying them to the ionacceleration region of the MS instrument. Mass spectrometry can then beperformed sequentially on “packets” of separated ion samples, ratherthan simultaneously on the bulk of the generated ions. In this manner,mass spectral information provided by the MS instrument may be spreadout in another dimension to thereby reduce the localized congestion ofmass information associated with the bulk ion analysis.

One known ion separation technique which may be used to pre-separate thebulk of the ions in time prior to MS analysis is ion mobilityspectrometry (IMS). As is known in the art, IMS instruments typicallyinclude a pressurized static buffer gas contained in a drift tube whichdefines a constant electric field from one end of the tube to the other.Gaseous ions entering the constant electric field area are acceleratedthereby and experience repeated collisions with the buffer gas moleculesas they travel through the drift tube. As a result of the repeatedaccelerations and collisions, each of the gaseous ions achieves aconstant velocity through the drift tube. The ratio of ion velocity tothe magnitude of the electric field defines an ion mobility, wherein themobility of any given ion through a high pressure buffer gas is afunction of the collision cross-section of the ion with the buffer gasand the charge of the ion. Generally, compact conformers, i.e. thosehaving smaller collision cross-sectional areas, have higher mobilities,and hence higher velocities through the buffer gas, than diffuseconformers of the same mass, i.e. those having larger collisioncross-sectional areas. Thus, ions having larger collision cross-sectionsmove more slowly through the drift tube of an IMS instrument than thosehaving smaller collision cross-sections, even though the ions havingsmaller collision cross-sections may have greater mass than those havinghigher collision cross-sections. This concept is illustrated in FIG. 2which shows drift times through a conventional IMS instrument for threeions, each having a different mass and shape (collision cross-section).As is evident from FIG. 2, the most compact ion 14 (which appears tohave the greatest mass) has the shortest drift time peak 16 ofapproximately 5.0 ms, the most diffuse ion 18 has the longest drift timepeak 20 of approximately 7.4 ms, and the ion 22 having a collisioncross-section between that of ion 14 and ion 18 (which also appears tohave the least mass), has a drift time peak 24 of approximately 6.1 ms.

Referring now to FIG. 3, an ion time-of-flight spectrum 26, obtainedfrom a known time-of-flight mass spectrometer, is shown plotted vs. iondrift time. In this figure, ions of different mass are dispersed overdifferent times of flight in the mass spectrometer. However, due to thelimited resolution of the mass spectrometer, ions are not completelyseparated in the spectrum, i.e. dots corresponding to different ionsoverlap. When compared with FIG. 6, which will be discussed more fullyin the DESCRIPTION OF THE PREFERRED EMBODIMENTS section, it is evidentthat different ions can be better resolved by an instrument thatseparates ions in two dimensions, namely ion mobility and ion mass.

Guevremont et al. have recently modified an existing IMS/MS instrumentto convert a quadrupole MS to a TOFMS [R. Guevremont, K. W. M. Siu, andL. Ding, PROCEEDINGS OF THE 44^(TH) ASMS CONFERENCE, (1996), Abstract].Ions are generated in the Guevremont et al. instrument via electrospray,and 5 ms packets are gated into the IMS instrument. The ion packetsproduced by the IMS instrument are passed through a small opening intoan ion acceleration region of the TOFMS.

While Guevremont et al. have had some experimental success in couplingan IMS instrument to a TOFMS instrument, their resulting instrumentationand techniques have several drawbacks associated therewith. For example,since the Guevremont et al. abstract discusses using 5 ms gate pulses toadmit ions into the IMS instrument, it is noted that the resultant IMSspectrum has low resolution with at least 5 ms peak widths. Secondly,because the drift tube and ion flight tube of the Guevremont et al.instrument are colinear, any spatial and temporal spread in an ionpacket leaving the IMS leads directly to a spatial and temporal spreadof ions in the ion acceleration region of the TOFMS. These twocharacteristics lead to poor mass resolution in the TOFMS. Thecombination of low resolution in the IMS and low resolution in the TOFMSmakes this instrument incapable of resolving complex mixtures. What istherefore needed is a hybrid IMS/TOFMS instrument optimized to resolvecomplex mixtures. Such an instrument should ideally provide foroptimization of the ion mobility spectrum as well as optimization of themass spectrum. Moreover, such a system should provide for an optimuminterface between the two instruments to thereby maximize thecapabilities of the TOFMS.

SUMMARY OF THE INVENTION

The foregoing drawbacks associated with the prior art systems discussedin the BACKGROUND section are addressed by the present invention. Inaccordance with one aspect of the present invention, a method ofgenerating ion mass spectral information comprises the steps ofgenerating a gaseous bulk of ions, gating at least a portion of the bulkof ions into an ion mobility spectrometer to thereby separate the bulkof ions in time to form a number of ion packets each having an ionmobility associated therewith, sequentially directing at least some ofthe ion packets into a mass spectrometer, continually activating themass spectrometer to thereby sequentially separate at least some of theion packets in time to form a number of ion subpackets each having anion mass associated therewith, and processing at least some of the ionsubpackets to determine mass spectral information therefrom.

In accordance with another aspect of the present invention, an apparatusfor generating mass spectral information from a sample source comprisesmeans for generating a gaseous bulk of ions from a sample source, an ionmobility spectrometer (IMS) having an ion inlet coupled to the means forgenerating a gaseous bulk of ions and an ion outlet, wherein the IMS isoperable to separate ions in time as a function of ion mobility, a massspectrometer (MS) having an ion acceleration region coupled to said ionoutlet of said IMS and an ion detector, wherein the MS is operable toseparate ions in time as a function of ion mass, and a computer operableto gate at least a portion of the gaseous bulk of ions into the ioninlet of the IMS and to continually pulse the ion acceleration region ofthe MS to thereby sequentially direct ions toward the ion detector.

In accordance with yet another aspect of the present invention, a methodof generating ion mass spectral information comprises the steps ofgenerating a gaseous bulk of ions, separating the gaseous bulk of ionsin time as a function of ion mobility, where two or more ions overlap inion mobility values, filtering out ions that have all but a desiredmass-to-charge ratio, sequentially separating in time the post-filteredions as a function of ion mass, and processing ions separated astwo-dimensional functions of ion mobility and ion mass to determine ionmass spectral information therefrom.

In accordance with a further aspect of the present invention, anapparatus for generating mass spectral information from a sample sourcecomprises means for generating a gaseous bulk of ions from a samplesource, an ion mobility spectrometer (IMS) having an ion inlet coupledto the means for generating a gaseous bulk of ions and an ion outlet,wherein the IMS is operable to separate ions in time as a function ofion mobility, an ion filter having a filter inlet coupled to the ionoutlet of the IMS and a filter outlet, wherein the ion filter isoperable to sequentially pass therethrough only ions having desiredmass-to-charge ratios, and a mass spectrometer (MS) having an ionacceleration region coupled to the filter outlet and an ion detector,wherein the MS is operable to sequentially separate in time ionsprovided thereto by the ion filter as a function of ion mass.

In accordance with still a further aspect of the present invention, amethod of generating ion mass spectral information comprises the stepsof generating a gaseous bulk of ions, separating the gaseous bulk ofions in time as a function of ion mobility, sequentially separating intime as a function of ion mass each of the ions separated in time as afunction of ion mobility, processing ions separated as two-dimensionalfunctions of ion mobility and ion mass to determine ion mass spectralinformation therefrom, repeating the generating and separating stepsfollowed by the step of sequentially fragmenting into daughter ions eachof the ions separated in time as a function of ion mobility, followed bythe sequentially separating and processing steps only if the initialprocessing step indicates that no two or more ions overlap in mobilityvalues. If, on the other hand, the initial processing step indicatesthat two or more ions overlap in mobility values, the method furtherincludes the step of filtering out ions that have all but a desiredmass-to-charge ratio, followed by repeating the generating andseparating steps, followed by the step of sequentially fragmenting intodaughter ions each of the ions separated in time as a function of ionmobility, followed by the sequentially separating and processing steps.Thereafter, the method further includes the step of repeating thefiltering step until all ions overlapping in ion mobility values havebeen processed.

One object of the present invention is to provide instrumentation forrapid analysis and sequencing of large biomolecules, as well as analysisof mixtures of organic and inorganic molecules.

Another object of the present invention is to provide an ion mobilityand time-of-flight spectrometer for composition, sequence and structuralanalysis of biomolecules.

Yet another object of the present invention is to optimize such aninstrument for sensitivity and resolution of both ion mobility and ionmass spectra.

Still another object of the present invention is to provide a techniquefor operating such an instrument in obtaining sequencing information.

These and other objects of the present invention will become moreapparent from the following description of the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a MALDI-TOF mass spectrum of cytochrome-c and lysozyme.

FIG. 2 is an IMS drift time distribution for three ions having differentcollision cross-sections.

FIG. 3 is a mass spectrum plotted against drift time illustrating thelimited resolution of a time-of-flight mass spectrometer.

FIG. 4 is a cross-section and schematic diagram of one embodiment of ahybrid ion mobility and time-of-flight mass spectrometer, in accordancewith the present invention.

FIG. 5 is a cross-section and schematic diagram of an alternateembodiment of a hybrid ion mobility and time-of-flight massspectrometer, according to the present invention.

FIG. 6 is a plot of ion time-of-flight vs. ion drift time foroligothymidine, utilizing the hybrid instrumentation of either FIG. 4 orFIG. 5.

FIG. 7A is a diagrammatic illustration of one preferred embodiment of anion source for use with any of the instrument configurations shown inFIGS. 4, 5 and 9.

FIG. 7B is a diagrammatic illustration of an alternate embodiment of anion source for use with any of the instrument configurations shown inFIGS. 4, 5 and 9.

FIG. 7C is a diagrammatic illustration of another alternate embodimentof an ion source for use with any of the instrument configurations shownin FIGS. 4, 5 and 9.

FIG. 8A is a plot of ion intensity vs. ion drift time for an IMSinstrument without an ion trap disposed between the ion source and theIMS instrument.

FIG. 8B is a plot of ion intensity vs. ion drift time for an IMSinstrument having an ion trap disposed between the ion source and theIMS instrument.

FIG. 9 is a block diagram illustration of an another alternateembodiment of an ion mobility and time-of-flight mass spectrometer, inaccordance with the present invention.

FIG. 10 is a partial cross-sectional diagram of yet another alternateembodiment of an ion source for use with any of the instrumentconfigurations shown in FIGS. 4, 5 and 9.

FIG. 11 is a cross-section of one preferred embodiment of the quadrupolemass filter illustrated in FIG. 9 as viewed along section lines 11—11.

FIG. 12 is a plot of ion intensity vs. mass-to-charge ratio illustratingoperation of the quadrupole mass filter of FIG. 11.

FIG. 13 is a flowchart illustrating one preferred embodiment of aprocess for conducting sequencing analysis using the instrumentconfiguration of FIG. 9, in accordance with the present invention.

FIG. 14 is composed of FIGS. 14A-14D and illustrates an example ionmass/mobility spectrum resulting from a first pass through the processillustrated in FIG. 13.

FIG. 15 is composed of FIGS. 15A-15D and illustrates an example ionmass/mobility spectrum resulting from a second pass through the processillustrated in FIG. 13.

FIG. 16 is composed of FIGS. 16A-16D and illustrates an example ionmass/mobility spectrum resulting from a third pass through the processillustrated in FIG. 13.

FIG. 17 is a block diagram illustrating alternative structuralvariations of the ion mobility and time-of-flight mass spectrometer ofthe present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill nevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations and furthermodifications in the illustrated devices, and such further applicationsof the principles of the invention as illustrated therein beingcontemplated as would normally occur to one skilled in the art to whichthe invention relates.

Referring now to FIG. 4, one preferred embodiment of a hybrid ionmobility and time-of-flight mass spectrometer instrument 30, inaccordance with the present invention, is shown. Instrument 30 includes,as its basic components, an ion source region 32 in communication withan ion mobility spectrometer 34, which itself is in communication with amass spectrometer 36. A computer 38 is provided for controlling at leastsome portions of the instrument 30 as well as for collecting ioninformation from mass spectrometer 36. Computer 38 is preferably apersonal computer (PC) of known construction having at least a known 386processor, although the present invention contemplates that computer 38may be any known computer, controller or data processor capable ofcontrolling instrument 30, as set forth in greater detail hereinafter,and of collecting and processing ion information from mass spectrometer36.

Preferably, mass spectrometer 36 is of the linear time-of-flight type,although the present invention contemplates that spectrometer 36 mayalternatively be a known reflectron time-of-flight mass spectrometer,multi-pass time-of-flight mass spectrometer or Fourier Transformion-cyclotron-resonance (FTICR-MS) mass spectrometer. In one preferredembodiment, the TOFMS 36 is configured to maximize mass resolution byminimizing the deleterious effects of initial ion position and initialion velocity distributions. Details of such a TOFMS configuration andoperation thereof are given in U.S. Pat. Nos. 5,504,326, 5,510,613 and5,712,479 to Reilly et al., all assigned to the assignee of the presentinvention, and the contents of which are all incorporated herein byreference.

Ion mobility spectrometer (IMS) 34 includes a drift tube 40 having a gasport 42 disposed adjacent to an ion exit end 44 of tube 40, wherein port42 is connected to a source of buffer gas 46. The flow rate of buffergas may be controlled by computer 38 via signal path 48, or mayalternatively be controlled by a manually actuated valve (not shown).Ion exit end 44 of drift tube 40 includes an endplate 43 attachedthereto, wherein endplate 43 defines an opening, or ion aperture, 45therethrough.

Drift tube 40 includes a number of guard rings 50 distributed along itsinner surface, wherein the guard rings 50 are interconnected byequivalent-valued resistors (not shown). The guard ring positioned mostadjacent to ion source region 32 is connected to a voltage source VS1 52via signal path 54, and source 52 is preferably controlled by computer38 via signal path 56, although the present invention contemplatescontrolling source 52 via a manual actuator (not shown). The drift tube40 defines a longitudinal axis 72 therethrough which will be referred tohereinafter as the drift tube axis 72. Voltage source 52 is preferablyset to a positive voltage to thereby establish a constant electric fielddirected along axis 72 in a direction indicated by arrow 55. Thoseskilled in the art will recognize that the importance of the guard ringand voltage source arrangement of the spectrometer 34 lies not in itsspecific structure, but in its ability to establish, as accurately aspossible, a constant electric field in the direction of arrow 55. Inthis sense, the present invention contemplates that any known structureor arrangement may be used to establish such an electric field withindrift tube 40 in the direction of arrow 55. It is to be understood,however, that a constant electric field in the direction of arrow 55 isestablished to accelerate positively charged ions toward tube end 44,and that such an electric field may be reversed to thereby acceleratenegatively charged ions toward tube end 44.

Drift tube 40 may optionally be surrounded by a variable temperaturehousing 58 which is connected to a variable temperature source 60 viapath 62, all of which are shown in phantom. In one embodiment, variabletemperature source 60 is a fluid holding tank and path 62 is a conduitleading to housing 58 which, in this case, is preferably sealed. Areturn conduit (not shown) is also connected to the fluid holding tankso that fluid from within the tank may be circulated through housing 58.The fluid within the fluid holding tank may be a heated or cooled gas orliquid such as, for example, liquid nitrogen. In an alternateembodiment, variable temperature source 60 is a known electricallyactuatable temperature controller, and path 62 comprises a pair ofelectrical conductors connected between the controller and housing 58.In operation, temperature controller 60 is operable to heat or coolhousing 58 as desired. Regardless of the particular embodiment ofhousing 58, source 60 and path 62, the present invention contemplatesthat source 60 may furthermore be controlled by computer 38 via signalpath 64.

Drift tube 40 is further surrounded by a housing 70 which defines a tubeend 66 covering an ion entrance end thereof, wherein tube end 66 definesan opening, or ion aperture, 68 therethrough, and an ion exit opening,or aperture, 84 adjacent to endplate 43. Preferably, ion optics 47 arepositioned between openings 45 and 84 to focus ions exiting opening 45into an ion acceleration region of TOFMS 36. Openings 45, 68 and 84 arepreferably bisected by drift tube axis 72. An ion source 74, which willbe described more fully hereinafter, is positioned within ion sourceregion 32 and is operable, preferably under the control of computer 38via a number, N, of signal paths 76, wherein N may be any positiveinteger, to direct ions within the spectrometer 34 via opening 68. Ionsentering drift tube 40 separate in time as a function of theirindividual mobilities, as discussed hereinabove, and are sequentiallydirected through opening 70 toward TOFMS 36.

Housing 70 includes a pump 80 for controlling the pressure of the buffergas. Preferably, pump 80 is a diffusion pump, the operation of which maybe controlled by computer 38 via signal path 82. Alternatively, pump 80may be manually controlled by a manual pump actuator (not shown). In anycase, pump 80 is operable to establish a desired pressure of the staticbuffer gas within drift tube 40. In accordance with known IMStechniques, the buffer gas within drift tube 40 may typically be setwithin the range of between approximately one and a few thousand Torr.

TOFMS 36 is preferably surrounded by a housing 126 that is attached toIMS 34. TOFMS 36 includes a first electrically conductive grid or plate86 connected to a second voltage source VS2 88 via signal path 90, whichis preferably controlled by computer 38 via signal path 92. A secondelectrically conductive grid or plate 94 is connected to a third voltagesource VS3 96 via signal path 98, which is preferably controlled bycomputer 38 via signal path 100. A third electrically conductive grid orplate 102 is connected to a fourth voltage source VS4 via signal path106, which is preferably controlled by computer 38 via signal path 108.Grids or plates 86, 94 and 102 define first and second ion accelerationregions therebetween as is known in the art, and which will be morefully described hereinafter. Those skilled in the art will recognizethat other known ion acceleration region structures may be used withTOFMS 36, such as, for example, positioning a fourth grid or platebetween grids or plates 94 and 102.

Grid or plate 102 has a plate surface attached to one end of a flighttube 110, the opposite end of which is attached to a surface of a fourthelectrically conductive grid or plate 112. An ion detector 116 isdisposed adjacent to grid or plate 112 with an air gap 114 definedtherebetween. Ion detector 116 is connected to a fifth voltage sourceVS5 118 via signal path 120, which is preferably controlled by computer38 via signal path 122. Ion detector 116 further has a signal outputconnected to computer 38 via signal path 124, whereby detector 116 isoperable to provide ion arrival time information to computer 38. Gridsor plates 86, 94, 102 and 112 are preferably arranged in juxtapositionwith each other such that all plate surfaces having greatest surfacearea are parallel with each other as well as to the surface of the iondetector 116, and are further preferably perpendicular to a longitudinalaxis 128 defined centrally through the flight tube 110, which willhereinafter be referred to as the flight tube axis 128.

TOFMS 36 further includes a pump 130 for controlling the vacuum of theTOFMS chamber defined by housing 126. Preferably, pump 130 is adiffusion pump, the operation of which may be controlled by computer 38via signal path 132. Alternatively, pump 130 may be manually controlledby a manual pump actuator (not shown). In any case, pump 130 is operableto establish a desired vacuum within housing 126 which may be set, inaccordance with know TOFMS operating techniques, to within the range ofbetween approximately 10⁻⁴ and 10⁻¹ Torr.

In the instrument 30 illustrated in FIG. 4, TOFMS 36 is preferablyarranged relative to IMS 34 such that the flight tube axis 128 isperpendicular to the drift tube axis 72. Moreover, TOFMS 36 ispreferably positioned relative to IMS 34 such that the drift tube axis72 and the flight tube axis 128 bisect within the first ion accelerationregion defined between grids or plates j86 and 94. In an alternativeconfiguration of TOFMS 36, grid or plate 94 may be omitted, and theTOFMS 36 need then be positioned relative to IMS 34 such that the drifttube axis 72 bisects the flight tube axis 128 within the ionacceleration region defined between grids or plates 86 and 102. Ineither case, TOFMS is preferably positioned relative to IMS 34 such thatthe drift tube axis 72 bisects the flight tube axis 128 approximatelycentrally within the region of interest.

In the operation of instrument 30, ions are generated by ion source 74,in accordance with one or more ion generation techniques describedhereinafter, and are supplied to IMS 34 via IMS inlet opening 68. Abuffer gas typically used in IMS instruments 34 is supplied to drifttube 40 via buffer gas source 46, wherein the buffer gas is regulated toa desired pressure via pump 80, buffer gas source 46 or a combinationthereof. Typically, the buffer gas is regulated to a pressure of betweenapproximately 1 and a few thousand Torr. Voltage source 52 supplies avoltage sufficient to generate a constant electric field along the drifttube axis in a direction indicated by arrow 55.

In accordance with known IMS 34 operation, ions entering IMS inletopening 68 travel through drift tube 40 toward IMS outlet opening 84,wherein the ions separate in time according to their individualmobilities. Ions having low mobility lag behind those having highermobility, wherein ion mobilities are largely a function of theircollision cross-sections. As a result, the more compact ions arrive atthe IMS outlet opening 84 more quickly than more diffuse ions. Thoseskilled in the art will recognize that the temperature of drift tube 40may also be controlled via variable temperature source 60 so that ionmobility analysis may be performed as a function of temperature.

TOFMS 36 is operable to accelerate ions from the space defined betweengrids or plates 86 and 94 toward a field-free flight tube 110, whereinthe ions separate in time according to their individual masses.Generally, ions having less mass will reach the detector 116 morequickly than those having greater mass. The detector 116 is operable todetect arrival times of the ions thereat and provide signalscorresponding thereto to computer 38 via signal path 124.

As set forth in greater detail in U.S. Pat. Nos. 5,504,326, 5,510,613and 5,712,479 to Reilly et al., which have been incorporated herein byreference, voltage sources VS2 88, VS3 96 and VS4 104 are typicallycontrolled by computer 38 to initially establish voltages at grids orplates 86, 94 and 102 that match the voltage level associated with IMS24 (which is set by voltage source VS1 52). Depending upon variousinstrument parameters, such as the length of flight tube 110, thedistances between grids or plates 88, 94, 102 and 112, and the distance114 between grid or plate 112 and detector 116, as well as estimates ofinitial ion position or initial ion velocity within the space definedbetween grids or plates 86 and 94, computer 38 is operable to controlsources 88, 96 and/or 104 to instantaneously increase the electric fieldbetween grids or plates 86, 94 and 102 to thereby create an ion drawoutelectric field therebetween which accelerates ions between these gridstoward flight tube 110. Preferably, the pulsed ion drawout electricfield is in a direction from grid or plate 86 toward flight tube 110 tothereby accelerate positively charged ions toward the flight tube 110.Those skilled in the art will recognize, however, that this electricfield may alternatively be reversed to accelerate negatively chargedions toward the flight tube 110.

In any event, ions within the space defined between grids or plates 86and 94 are accelerated by the pulsed ion drawout electric field to thespace defined between grids or plates 94 and 102. Due to the fact thations entering the region defined between grids or plates 86 and 94 alongaxis 72 have a narrow spatial distribution, due to focusing of the ionsinto this region via ion optics 47, and a small velocity component alongaxis 128, it is possible to choose the pulsed voltage applied to gridsor plates 86 and/or 94 in such a way as to obtain sharp TOFMS peaks. Thegoal of the pulsed ion drawout electric field and the subsequentacceleration of the ions between grids or plates 94 and 102 is toprovide all ions reaching grid or plate 102 with substantially the samekinetic energy. The flight tube 110 has no electric field associatedtherewith so that the ions drift from grid or plate 102 toward detector116, wherein the ions separate in time as a function of their individualmasses as described hereinabove. Computer 38 typically controls voltagesource VS5 118 to supply a voltage thereto during detection times tothereby increase the gain of detector 116 as is known in the art. Pump130 controls the vacuum within TOFMS 36, and pump 130 is preferablycontrolled by computer 38 via signal path 132. TOFMS 36 is typicallyoperated between 10⁻⁴ and 10⁻¹⁰ Torr.

In the embodiment 30 of the hybrid IMS/TOFMS instrument illustrated inFIG. 4, drift tube axis 72 preferably bisects the space defined betweengrids or plates 86 and 94 of TOFMS 36, and is perpendicular to flighttube axis 128. The present invention alternatively contemplatesarranging TOFMS 36 relative to IMS 34 such that the drift tube axis 72passes between grids or plates 86 and 94 perpendicular to flight tubeaxis 128, but at some other known distance relative to either of thegrids or plates 86 and 94. In either case, the foregoing structuralpositioning of TOFMS 36 relative to IMS 34 provides advantages overnon-perpendicular arrangements of the drift tube axis 72 relative to theflight tube axis 128. For example, such a perpendicular arrangementensures that ion packets entering the ion acceleration region definedbetween grids or plates 86 and 94 from IMS 34 will have constant andrelatively well defined initial ion positions as they traveltherebetween along axis 72. As discussed briefly hereinabove, ion optics47 focus ions into the ion acceleration region to thereby minimizespatial distribution of the ions. Moreover, since axis 72 is parallelwith grids or plates 86 and o4, ion position with respect to axis 128will remain relatively constant. This feature provides for the abilityto accurately estimate initial ion position within the ion accelerationregion defined between grids or plates 86 and 94, to thereby allow amore accurate estimation of the pulsed ion drawout electric fielddiscussed above.

Preferably, computer 38 controls the generation of ions from ion source74, as will be discussed in greater detail hereinafter, so that computer38 has knowledge of the times at which ions were introduced into IMS 34,hereinafter referred to as ion introduction events. The computer 38 isthen operable to control voltage sources 88 and 96 to repeatedly providethe pulsed ion drawout field some number of times for every ionintroduction event. In one embodiment, a pulsed ion drawout field isrepeatedly provided 512 times for every ion introduction event. Thoseskilled in the art will recognize that the number of pulsed ion drawoutfields provided for every ion introduction event is directlyproportional to the ultimate resolution of the instrument 30. As thispulsed operation relates to some of the advantages of the perpendicularpositioning of TOFMS 36 relative to IMS 34, such an arrangementminimizes the possibility that all or part of any one ion packet willtravel through the TOFMS 36 unprocessed. Due to the direction of travelof the ion packets relative to the grids or plates 86 and 94, and alsoto the pulsed nature of the ion drawout electric field, the TOFMS 36will have multiple chances to accelerate each ion packet toward detector116 as they travel along axis 72. As such, the instrument 30 isconfigured to provide for maximum ion throughput to detector 116.

Referring now to FIG. 5, an alternate embodiment of a hybrid ionmobility and time-of-flight mass spectrometer 150, in accordance withthe present invention, is shown. Spectrometer 150 is similar in manyrespects to spectrometer 30 shown in FIG. 4 and described hereinabove,and like components are therefore identified with like numbers.Discussion of the common components, as well as the basic operation ofIMS 34 and TOFMS 36′, will therefore not be repeated for brevity's sake.

Unlike instrument 30 of FIG. 4, the TOFMS 36′ of instrument 150 ispositioned relative to IMS 34 such that the drift tube axis 72 alsodefines the flight tube axis of TOFMS 36′. Alternatively, TOFMS 36′could be arranged relative to IMS 34 with any orientation such that thedrift tube axis 72 is non-perpendicular to the flight tube axis. In anysuch orientation, the initial positions of the ion packets within thespace defined between grids or plates 86′ and 94 either cannot beestimated with any degree of accuracy (as in the orientationillustrated) or changes as the ion packets travel along axis 72 (as inany non-perpendicular arrangement). Moreover, in any such orientation,it is difficult to estimate when, relative to an ion introduction event,the ion packets will arrive within the space defined between grids orplates 86′ and 94, and the timing of the pulsed ion drawout electricfields is thus difficult to predict. As a result, it is likely that thetiming of the pulsed ion drawout electric fields will be inaccurate sothat ions may be lost within the TOFMS 36′ and/or the mass resolution ofthe TOFMS 36′ will be adversely affected.

In order to address the foregoing problems associated withnon-perpendicular positioning of the TOFMS 36′ relative to the IMS 34,which are the same problems associated with the Guevremont et al. systemdiscussed hereinabove in the BACKGROUND section, instrument 150 isprovided with an ion trap 152 operatively positioned between the ionoutlet opening 84 of IMS 34 and the space defined between grids orplates 86′ and 94. In the embodiment illustrated in FIG. 5, grid orplate 86′ defines an ion inlet opening 178 therethrough which is alignedalong axis 72 with ion outlet opening 84 of IMS 34. In othernon-perpendicular arrangements of TOFMS 36′ relative to IMS 34, ioninlet opening 178 may not be required since ions may enter the spacebetween grids or plates j86′ and 94 in the same manner as discussed withrespect to the embodiment 30 illustrated in FIG. 4.

In any event, ion trap 152 is preferably a known quadrupole ion traphaving a first endcap 154, a center ring 162 and a second endcap 170.Each of the endcaps 154 and 170 define apertures therethrough whichalign with axis 72. In this configuration, ion trap 152 confines ionstherein to a small volume in its center which is in alignment with theion inlet opening to TOFMS 36′. First endcap 154 is connected to avoltage source VS6 156 via signal path 158, which is itself connected tocomputer 38 via signal path 160. Center ring 162 is connected to avoltage source VS7 164 via signal path 166, which is itself connected tocomputer 38 via signal path 168, and second endcap 170 is connected to avoltage source VS8 172 via signal path 174, wherein source 172 isconnected to computer 38 via signal path 176. Preferably, sources 156and 172 are operable to produce DC voltages and source 164 is operableto produce AC voltages in the RF range.

In operation, computer 38 controls sources 156 and 172 to bias endcaps154 and 170 such that ions exiting ion outlet opening 84 of IMS 34 havejust enough energy to enter the opening defined in the first endcap 154.Once therein, the ions collide with buffer gas leaking out of opening 84into the trap 152, and lose sufficient energy thereby so that the RFvoltage on center ring 162 is operable to confine the ions within thetrap 152. The confined ions undergo further collisions inside the trap152 which causes the ions to correspondingly experience further energyloss, resulting in a concentration of the ions toward the center of ring162 due to the RF voltage thereon. As long as the voltages on endcaps154 and 170 and center ring 162 are maintained, ions may enter the trap152 and collect therein. Ions are ejected out of the trap 152 by turningoff the RF voltage on center ring 162 and applying an appropriate DCpulse to one of the endcaps 154 or 170. For example, to eject acollection of positively charged ions from trap 152, either the voltageon endcap 154 may be pulsed above that present on endcap 170 or thevoltage on endcap 170 may be pulsed below that present on endcap 154. Ingeneral, the magnitude of the RF field applied to the center ring viasource 164, as well as any DC voltage included therein, may be varied tothereby select ions of any desired mass to charge ratio to be collectedby ion trap 152. Ions of all mass to charge ratios, or ions of anyparticular mass to charge ratio, may be selectively collected within iontrap 152 through proper choice of DC level and RF peak magnitudeprovided by voltage source 164.

As it relates to the present invention, the ion trap 152 is controllableby computer 38 to periodically eject the collected ion packetstherefrom, hereinafter referred to as an ion ejection event, so as toprovide for a more accurate estimate of initial ion position within thespace defined between grids or plates 86′ and 94. Since the computer 38controls the time at which a packet of collected ions is ejected fromion trap 152, the time at which the ion packet arrives at a specifiedposition in the space defined between grids or plates 86′ and 94 can beaccurately estimated. Knowing the approximate time, relative to the ionejection event, at which the ion packet arrives at the specifiedposition between grids or plates 86′ and 94, computer 38 may moreaccurately estimate appropriate timing for applications of the pulsedion drawout electric field to thereby provide for maximum massresolution as discussed hereinabove. Moreover, providing for a moreaccurate estimate of the timing of the pulsed ion drawout electricfields reduces the likelihood that ion packets, or at least portionsthereof, will be lost within the TOFMS 36′.

In the operation of instrument 150, IMS 34 is operable to providepackets of ions, which are separated in time as a function of ionmobility, to TOFMS 36′ via ion outlet opening 84. Computer 38 controlsion trap 152 to collect the various ion packets therein one at a time,and eject each collected ion packet therefrom at periodic intervals. Theejected ions enter the space defined between grids or plates 86′ and 94as discussed hereinabove, and computer 38 is operable to computerappropriate times at which to apply the pulsed ion drawout electricfields based on the timing of the ion ejection events. The TOFMS 36′ isthereafter operable as described hereinabove to produce mass spectruminformation.

Referring now to FIG. 6, a plot 190 of ion flight time vs. ion drifttime for an oligothymidine sample is shown, wherein the data shown isproducible via either instrument embodiment 30 or 150. As compared tothe plot of FIG. 3, it is apparent that the hybrid ion mobility andtime-of-flight mass spectrometer of the present invention is operable toresolve structural information of molecules in two substantiallyorthogonal dimensions. For each drift time, corresponding to arrival inthe TOFMS of a corresponding ion packet, the instrument of the presentinvention is operable to resolve a number of times-of-flight,corresponding to a number of mass to charge ratios. The plot 190 of FIG.6 thus illustrates that the total resolving power of instrument 30 isdrastically better than that achievable via an IMS or TOFMS alone. Thistechnique dramatically reduces the problem of congestion of massspectra, due to mass peak overlap, in obtaining sequence information forlarge biomolecules (in excess of 50 residues). The present inventionthus provides an instrument for composition, sequence and structuralanalysis of biomolecules which does not suffer from drawbacks associatedwith prior art systems discussed in the BACKGROUND section.

Referring now to FIG. 7A, one preferred embodiment 74′ of an ion source74 for either of the instrument embodiments of FIGS. 4 and 5, is shown.Embodiment 74′ includes a chamber 200 having a sample 202 mountedtherein and an optical window 206 extending therefrom. A radiationsource 204 is electrically connected to computer 38 via signal path 76A,and is configured to direct radiation through optical window 206 tothereby irradiate sample 202. Chamber 200 may include a conduitextending therefrom to a pump 208 which may be controlled by computer 38via signal path 76B.

Ion source 74′ is a known MALDI arrangement wherein radiation source204, preferably a laser, is operable to desorb gaseous ions from asurface of the sample 202. Computer 38 is operable to control activationtimes of laser 204 to thereby control sample ionization events. Thedesorbed ions are directed by the internal structure of chamber 202 toion inlet opening 68 of IMS 34. The sample 202 may, in accordance withthe present invention, be a biomolecule of any size such as DNA, RNA,any of various proteins, carbohydrates, glycoconjugates, and the like.Pump 208 may be controlled to pressurize chamber 208 to thereby conducthigh pressure MALDI analysis as is known in the art.

Referring now to FIG. 7B, an alternate embodiment 74″ of an ion source74 for either of the instrument embodiments of FIGS. 4 and 5, is shown.Embodiment 74″ includes a liquefied sample 220 having a spray hose ornozzle 222 extending toward an opening defined in a desolvation region226. Actuation of the spray nozzle 222 may be manually controlled, as isknown in the art, or may be controlled by computer 38 via signal path76C. Desolvation region 226 is connected to computer 38 via signal path76C′, and is operable to convert charged sample droplets suppliedthereto via nozzle 222 into gaseous ions and supply these ions to a ionoptics member 228. Optics member 230 is operable to focus the gaseousions and direct them into ion inlet opening of IMS 34. Ion source region32 includes a conduit extending therefrom to a pump 232 which may becontrolled by computer 38 via signal path 76D.

Ion source 74″ is a known electrospray ionization (ESI) arrangementoperable to convert a liquefied solution containing the sample togaseous ions. Computer 38 is operable to control activation times ofdesolvation region 226 to thereby control sample ionization events. Pump232 is operable to pressurize the ion source region 32 as is known inthe art, and the desolvation region 226 is operable convert theliquefied solution to gaseous ions. The sample source 220 may, inaccordance with the present invention, include a solution containing abiomolecule of any size such as DNA, RNA, any of various proteins,carbohydrates, glycoconjugates, and the like.

Referring now to FIG. 7C, another alternate embodiment 74′″ of an ionsource 74 for either of the instrument embodiments of FIGS. 4 and 5, isshown. Embodiment 74′″ includes a sample source 236, which may be eitherof the foregoing sample sources 74′ or 74″ illustrated in FIGS. 7A or7B, and which may be controlled as described hereinabove by computer 38via a number, M, of signal paths 76E, wherein M may be any integer lessthan N (see FIGS. 4 and 5).

Ion source 74′″ further includes an ion trap 152 positioned between ionsource 236 and the ion inlet opening 68 of IMS 34. Ion trap 152 ispreferably a known quadrupole ion trap identical to that shown in FIG. 5and described hereinabove. A detailed discussion of the operation of iontrap 152 therefore need not be repeated here. Endcap 154 is connected toa voltage source VS9 238 via signal path 240, center ring 162 isconnected to a voltage source VS10 242 via signal path 244 and endcap170 is connected to a voltage source VS11 246 via signal path 248. VS9,VS10 and VS11 are each connected to computer 38 via signal paths 76F,76G and 76H, respectively. Computer 38 is operable to control VS9, VS10and VS11 identically as described with respect to VS6, VS7 and VS8,respectively, of FIG. 5.

In operation, computer 38 is operable to control ion trap 152, in amanner similar to that described hereinabove, to collect a bulk of ionstherein and selectively eject the collected ions therefrom toward ioninlet opening 68 of IMS 34. As is known in the art, the peak resolutionof an ion mobility instrument, such IMS 34, is limited by the length ofthe input pulse of ions into the instrument. Generally, mobility peakscannot be resolved any better than the time length of the input ionpulse. A drawback particularly associated with the use of ESI is thatthe input ion pulse width must typically be at least 50 μs in order toproduce enough ions for analysis. However, with the ion sourcearrangement 74′″ shown in FIG. 7C, computer 38 is operable to collect alarge number of ions within ion trap 152 prior to pulsing the ions intothe IMS 34. With a sufficient number of ions collected in ion trap 34,the only limitation on the ion input pulse length, and hence theresolution capability of IMS 34, is the time required to open and closeion trap 152. With existing ion traps, the ion input pulse lengths maybe reduced to less than 1.0 μs in duration.

FIGS. 8A and 8B show a comparison of ion mobility distributions for amaltotetraose sample, wherein the spectrum 250 of FIG. 8A was producedusing an ESI source similar to that shown in FIG. 7B, with 100,083 inputpulses of 20 μs duration. The spectrum 252 of FIG. 8B was produced usingthe same ESI source as that used for FIG. 8A along with an ion trap,such as ion trap 152 shown in FIG. 7C, with 4003 pulses of 1 μsduration. Compared to spectrum 250, spectrum 252 has a 4-5 timesincrease in signal strength, an increase in resolution by a factor ofapproximately 20 and an increase in signal-to-noise ratio by a factor ofapproximately 20 as well.

Referring again to FIG. 7C, ion trap 152 may be used with any known iongeneration source to increase not only the resolution and sensitivity ofIMS 34 along, but also the resolution and sensitivity of either hybridinstrument 30 or 150 of FIGS. 4 and 5.

It is to be understood that either embodiment of the hybrid ion mobilityand time-of-flight mass spectrometer shown and described herein iscapable of operation in a number of different operational modes. Forexample, the structure and operation of the various embodiments of thepresent invention have been described herein according to a first modeof operation wherein ions of relatively low energy are generated andinjected into the hybrid instrument, from which structural informationrelating to the ions can be obtained.

In a second mode of operation, such ions could be injected into thehybrid instrument at higher energies, wherein high energy collisionswith the buffer gas within the IMS 34 result in ion fragmentation. Insuch a case, the ion fragments, separated in time as a function of theirmobilities, would be supplied to the TOFMS portion of the instrument,wherein mass spectra information of the various fragments could beobtained for sequencing analysis. Alternatively, fragmentation of ionsfor such analysis may be accomplished via any of a number of other knowntechniques. Examples of such known alternative ion fragmentationtechniques include enzyme degradation fragmentation,photo-fragmentation, thermal dissociation such as by heating drift tube40 via control of variable temperature source 60, electron impactdissociation, surface induced dissociation, and blackbody infraredradiation induced dissociation.

In a third mode of operation, ions of only a particular mass could beprocessed by the hybrid instrument. One way of generating ions of only aparticular mass is to adjust the peak amplitude and/or DC voltage of thecenter ring voltage source of an ion trap positioned prior to the IMS34. By properly adjusting this voltage, ion trap 152 may be configuredto store therein only ions having a particular mass to charge ratio. Inthis manner, the ion trap 152 is controlled to act as an ion filter.Another way of analyzing ions of only a particular mass is to provide anion trap 152 between the IMS 34 and TOFMS 36, and controlling the iontrap 152 as just discussed to filter out ions having undesirable mass tocharge ratios.

In a fourth mode of operation, high energy ions of only a particularmass are introduced into the IMS 34. Therein, these ions undergofragmentation, and such fragments could then be further processed by theTOFMS 36 as discussed above.

Referring now to FIG. 9, one preferred embodiment of an ion mobility andmass spectrometer instrument 300 that is particularly well suited forconducting sequencing analysis in a manner similar to that justdescribed hereinabove with respect to the second mode of operation, inaccordance with the present invention, is shown. Several of thecomponents of instrument 300 are identical to those shown and describedwith respect to FIGS. 4 and 5, and some of the structural andoperational details thereof will accordingly be omitted here forbrevity. For example, instrument 300 includes an ion source 32operatively connected to an ion mobility spectrometer (IMS), wherein IMS34 includes a source of buffer gas 46 that is controllable via operationof a pump 80 as described hereinabove. Instrument 300 further includes amass spectrometer (MS) 36, preferably a time-of-flight mass spectrometer(TOFMS), that is configured to receive ions from IMS 34 as describedhereinabove. In this embodiment, however, the drift tube axis of IMS 34(not shown in FIG. 9) and the flight tube axis of TOFMS 36 (not shown inFIG. 9) may be arranged at any desired angle with respect to each other.It has been determined through experimentation that fornon-perpendicular configurations of IMS 34 relative to TOFMS 36 (i.e.,configurations other than that illustrated in FIG. 4), an ion trap 152(see FIG. 5) is not required as described hereinabove if the ionacceleration region (between grids 86, 94 and 102) of TOFMS 36 iscontinually activated or pulsed. In other words, ions need not becollected in an ion trap 152 for timing purposes if the ion accelerationregion of TOFMS 36 is continually pulsed in a free-running operationalmode. Accordingly, ion trap 152 may be omitted from any perpendicular ornon-perpendicular configurations of the IMS drift tube axis relative tothe TOFMS flight tube axis, although the present invention contemplatesthat such an ion trap 152 may optionally be used in such configurationsas desired, wherein trap 152 may be positioned adjacent to the entranceof TOFMS 36.

Instrument 300 further includes a computer 310 having a memory 312.Computer 310 is preferably operable to control the flow rate of buffergas #1 within buffer gas source 46 via signal path 48, and is furtherpreferably operable to control pump 80 of IMS 34 via signal path 82 anda vacuum pump 130 of TOFMS 36 via signal path 132, as describedhereinabove. Computer 310 is also operable to control ion source 32 viaa number, N, of signal paths 76, wherein N may be any integer, and isfurther operable to receive ion detection signals from TOFMS 36 viasignal path 124 and process such signals to produce two-dimensional ionspectra; e.g. ion mass vs. ion mobility, as described hereinabove.

Instrument 300 includes a number, J, of voltage sources 314 ₁-314 _(j)connected to computer 310 via signal paths 316 ₁-316 _(j). Voltagesources 314 ₁-314 _(j) are operatively connected to IMS 34 viacorresponding signal paths 318 ₁-318 _(j). In operation, computer 310 isoperable to control voltage sources 314 ₁-314 _(j) to thereby controlthe operation of IMS 34 as described hereinabove. Instrument 300 furtherincludes another number, M, of voltage sources 330 ₁-330 _(M) connectedto computer 310 via signal paths 332 ₁-332 _(M). Voltage sources 330₁-330 _(M) are operatively connected to TOFMS 36 via correspondingsignal paths 334 ₁-334 _(M). In operation, computer 310 is operable tocontrol voltage sources 330 ₁-330 _(M) to thereby control the operationof TOFMS 36 as described hereinabove.

The components of instrument 300 described thus far with respect to FIG.9 are identical to previously described components of the instruments 30and/or 150 of FIGS. 4 and 5. Unlike instruments 30 and 150, however,instrument 300 further includes a quadrupole mass filter 302 having anion inlet coupled to the ion outlet of IMS 34 and an ion outlet coupledto an ion inlet of a collision cell 304 of known construction. An ionoutlet of collision cell 304 is coupled to an ion inlet of TOFMS 36;i.e., to the ion acceleration region defined between plates or grids 86and 94 of TOFMS as shown in FIGS. 4 and 5. Collision cell 304 includes asource of buffer gas 306, wherein the flow rate of buffer gas #2 iscontrolled by computer 310 via signal path 307, preferably in a mannerdescribed hereinabove with respect to the computer control of the buffergas source 46 of FIG. 4. Alternatively, buffer gas source 306 may beomitted and buffer gas source 46 may be configured to provide buffer gas#1 to cell 304 via conduit 305 as shown in phantom in FIG. 9. Collisioncell 304 further includes a pump 308 of known construction, theoperation of which is controlled by computer 310 via signal path 309. Asis known in the art, pump 308 may be controlled to establish andmaintain a desired quantity of buffer gas within collision cell 304, andmay further be controlled to purge cell 304 of buffer gas.Alternatively, structure 308 may represent a manually actuatable orcomputer controlled valve. In this case, valve 308 may be controlled toestablish and maintain a desired quantity of buffer gas #2 withincollision cell 304, or may alternatively be controlled to establish andmaintain a desired quantity of buffer gas #1 within the quadrupole massfilter 302 and collision cell 304.

A number, K, of voltage sources 320 ₁-320 _(K) are provided, wherein Kmay be any integer, and wherein control inputs of sources 320 ₁-320 _(K)are connected to computer 310 via corresponding signal paths 322 ₁-322_(K). Outputs of voltage sources 320 ₁-320 _(K) are operativelyconnected to the quadrupole mass filter (QMF) 302, in a manner to bedescribed more fully hereinafter with respect to FIGS. 11 and 12, viacorresponding signal paths 324 ₁-324 _(K). A number, L, of voltagesources 326 ₁-326 _(L) are provided, wherein L may be any integer, andwherein control inputs of sources 326 ₁-326 _(L) are connected tocomputer 310 via corresponding signal paths 328 ₁-328 _(L). Outputs ofvoltage sources 326 ₁-326 _(L) are operatively connected to thecollision cell 304 in a known manner via corresponding signal paths 329₁-329 _(L).

Referring now to FIG. 10, a cross-section of another preferred structureof the ion source 32 for use with any of the instruments illustrated inFIGS. 4, 5 and 9, in accordance with the present invention, is shown.Ion source 32 includes an ion source chamber 350 separated from an ioncollection chamber 354 by a wall or partition 355. Ion source chamber350 includes a port having a conduit 352 connected thereto, whereinconduit 352 is preferably connected to a pump or valve of knownconstruction for changing gas pressure within region 350. An ion source74 is disposed within region 350, wherein source 74 may be any of theion sources 74′, 74″ or 74′″ described hereinabove with respect to FIGS.7A-7C, and/or any combination thereof. Wall or partition 355 includes anaperture 353 therethrough that is aligned with an ion outlet of ionsource 74 and is also preferably aligned with a longitudinal axis of thedrift tube 40 of IMS 34, wherein aperture 353 defines an ion inlet toion collection chamber 354. An electrically conductive grid, or seriesof vertically or horizontally parallel wires, 356 (hereinafter “grid”)is positioned across the ion inlet aperture 68 of IMS 34, wherein grid356 is connected to one of the voltage sources 314 ₁ via signal path 318₁. Computer 310 is operable to control the voltage of grid 356, as isknown in the art, to thereby permit and inhibit entrance of ions intoIMS 34. For example, computer 310 is operable to inhibit entrance ofions into IMS 34 by activating voltage source 314 ₁ to thereby causeions in the vicinity of grid 356 to be attracted thereto and neutralizedupon contact. Conversely, computer 310 is operable to permit entrance ofions into IMS 34 by deactivating voltage source 314 ₁ to thereby permitpassage of ions therethrough. Alternatively, the ion gating function maybe accomplished by a voltage source 320 ₂ connected to guard rings 50via signal path 318 ₂, wherein computer 310 is operable to controlsource 320 ₂ to attract ions to guard rings 50 when it is desirable toinhibit ions from traveling through drift tube 40. In this case, grid356 and voltage source 320 ₁ may be omitted from FIG. 10. Alternativelystill, the ion gating function may be accomplished by impressing avoltage across aperture 68 to thereby create an electric fieldtherebetween. In this case, computer 310 is operable to control thevoltage across aperture 68 to divert ions toward guard rings 50 when itis desirable to inhibit ions from traveling through drift tube 40. Thoseskilled in the art will recognize that any known technique for pulsingions from ion collection chamber 354 through ion inlet aperture 68,including for example any known electrical, mechanical and/or electromechanical means, may be used, and that any such technique falls withinthe scope of the present invention.

In any case, the ion collection chamber 354 is functionally similar tothe ion trap 152 of FIG. 7C in that it provides for the collection of alarge quantity of ions generated by ion source 74 prior to entrance intoIMS 34. Through appropriate control of ion source 74 and grid 356 orequivalent, the quantity of ions entering IMS 34 may thus becorrespondingly controlled.

Referring now to FIG. 11, a cross-section of the quadrupole mass filter(QMF) 302, as viewed along section lines 11—11 of FIG. 9, is shown. QMF302 includes four electrically conductive rods or plates 360, 362, 364and 366 that are preferably disposed equidistant from a longitudinalaxis 365 extending through QMF 302. Two of the opposing rods 360 and 362are electrically connected to voltage source 320 ₁ via signal path 324₁, wherein source 320 ₁ has a control input connected to computer 310via signal path 322 ₁. Signal path 324 ₁ is connected to a signal phaseshifter 366 of known construction via signal path 368, wherein a signaloutput of phase shifter 366 is electrically connected to the remainingtwo opposing rods 364 and 366. Computer 310 is operable to controlvoltage supply 320 ₁, which is preferably a radio frequency (RF) voltagesource, to thereby control the RF voltage applied to rods 360 and 362.Phase shifter 366 is preferably operable to shift the phase of the RFvoltage on signal path 368 by 180° and apply this phase shifted RFvoltage to signal path 324 ₂. Those skilled in the art will recognizethat phase shifter 366 may alternatively be replaced with a second RFvoltage source that is controllable by computer 310 to produce an RFvoltage identical to that produced by source 320 ₁ except shifted inphase by 180°. In any case, signal paths 324 ₁ and 324 ₂ areelectrically connected to voltage source 320 ₂ via signal paths 324 ₃and 324 ₄ respectively, wherein source 320 ₂ has a control inputconnected to computer 310 via signal path 322 ₂. Voltage source 320 ₂ ispreferably a DC voltage supply controllable by computer 310 to therebyimpress a DC voltage between rod pairs 360/362 and 364/366.

In the operation of QMF 302, the RF voltages applied to rods 360-366alternately attract ions to rod pairs 360/362 and 364/366, wherein thisattraction increases with decreasing ion mass-to-charge ratio (m/z).Below some threshold m/z value (i.e., lighter ions), the ions come intocontact with one of the rods 360-366 and are accordingly neutralized orejected. The m/z value below which ions are neutralized is determined bythe strength and frequency of the RF signal as is known in the art. TheDC voltage applied to rods 360-366 similarly attracts ions theretowherein this attraction increases with increasing m/z values. Above somethreshold m/z value (i.e., heavier ions), the ions come into contactwith one of the rods 360-366 and are accordingly neutralized. The m/zvalue above which ions are neutralized is determined by the strength ofthe DC signal as is known in the art. Referring to FIG. 12, a plot 370of ion intensity at the ion outlet of QMF 302 is shown demonstratingthat the RF and DC voltages applied to rods 360-366 result in passagethrough QMF 302 only of ions having m/z values above a minimum m/z valuem/z₁ and below a maximum m/z value m/z₂. QMF 302 thus acts as a bandpassfilter wherein the pass band of m/z values is controlled via computer310 by controlling the operating strength and frequency of the RFvoltage supply 320 ₁ and by controlling the operating strength of the DCvoltage supply 320 ₂. In accordance with an important aspect of thepresent invention, computer 310 is operable, under certain operatingconditions, to control the m/z values of ions being passed from IMS 34to the collision cell 304 as will be descried in greater detailhereinafter.

The collision cell 304 is of known construction, and the filling andpurging of buffer gas therein/therefrom is preferably controlled bycomputer 310 in a known manner. Alternatively, the filling and purgingof cell 304 may be manually controlled via known means. In either case,when cell 304 is filled with buffer gas, ions provided thereto by QMF302 undergo collisions with the buffer gas and fragmentation of parentions into a number of daughter ions results as is known in the art. In apreferred embodiment, the internal structure of the collision cell 304is similar to that of the quadrupole mass filter illustrated in FIG. 11except that collision cell 304 includes eight rods (poles) rather thanfour, and is accordingly referred to as an octopole collision cell. Atleast one of the voltage sources 326 ₁-326 _(L) is preferably a RFvoltage source connected between two pairs of four opposing poles,wherein computer 310 is operable to control the RF voltage source tothereby concentrate ions centrally therein and provide a low-losschannel or pipe between QMF 302 and MS 36. The buffer gas for cell 304may be, for example, Argon, Helium or Xenon, although the presentinvention contemplates using other gases provided to cell 304 via source306 or 46 as described hereinabove. The present invention contemplatesthat collision cell 304 may alternatively be configured in accordancewith any desired trapping multiple (e.g., quadrupole, hexapole, etc.).Alternatively still, collision cell 304 may me configured as anon-trapping gas collision cell. In any event, those skilled in the artwill recognize that the importance of any such collision cellarrangement lies in its ability to provide for fragmentation of enteringparent ions into daughter ions.

Referring now to FIG. 13, one preferred embodiment of a process 400 forconducting sequencing analysis using the instrument 300 illustrated inFIG. 9, in accordance with the present invention, is shown. Process 400begins at step 402 where a counter variable A is set equal to anarbitrary initial number (e.g., 1). Thereafter at step 404, collisioncell 304 is purged of buffer gas either manually or under the control ofcomputer 310 in a known manner. It is to be understood, however, that ifno buffer gas initially exists in cell 304, step 404 may be avoided.Thereafter at step 406, computer 310 is operable to control QMF 302 soas to pass ions having any m/z value therethrough. In one embodiment,computer 310 is operable to execute step 406 by deactivating voltagesources 320 ₁ and 320 ₂ to thereby operate QMF 302 in an all-passoperational mode; i.e., such that QMF 302 passes ions having all m/zvalues therethrough.

Process 400 continues from step 406 at step 408 where computer 310 isoperable to activate ion source 74 to thereby begin the generation ofions from a suitable sample source. Thereafter at step 410, controlcomputer 310 is operable to pulse ion gate 356 (FIG. 10) for apredetermined duration to thereby permit entrance of a gaseous bulk ofions from collection chamber 354 into IMS 34, and to continually pulsethe ion acceleration region of MS 36, as described hereinabove, tothereby operate MS 36 in a free running mode. Those skilled in the artwill recognize that when using embodiments of ion source 32 other thanthat shown in FIG. 10 (e.g., those of FIGS. 7A and 7B), steps 408 and410 may be combined such that computer 310 is operable to activate theion source and supply a gaseous bulk of ions to IMS 34 in a single step.In any case, process 400 continues from step 410 at step 412 where aspectrum of ion flight times (i.e., ion mass) vs. ion drift times (i.e.,ion mobilities) resulting from passage of ions through IMS 34 and MS 36,as described hereinabove, is observed.

Referring now to FIGS. 14A-14D, a graphical example of steps 410 and 412is illustrated. Signal 450 of FIG. 14A represents the voltage at iongate 356, wherein computer 310 is operable to pulse gate 356 to aninactive state for a predetermined duration at step 410 to therebypermit entrance of a bulk of gaseous ions into IMS 34. Signal 452 ofFIG. 14B represents the voltage at the ion acceleration region of TOFMS36, wherein computer 310 is operable to pulse the ion accelerationregion in a free running manner at step 410 to thereby periodicallyaccelerate ions or parts of ions toward the ion detector. A typicalvalue for the duration of deactivation of ion gate signal 450 is 100 μs,a typical value for the duration of activation of the TOFMS signal 452is 3 μs, and a typical value for the time between TOFMS signalactivation is 100 μs. However, the present invention contemplates othervalues for the foregoing signal durations, and it will be understoodthat the actual signal durations used will typically be dictated by manyfactors including sample type, analysis mode, information sought and thelike. In any case, signal 454 of FIG. 14C represents the activationstate of QMF 302, wherein computer 310 is operable throughout steps 410and 412 to maintain QMF 302 in an inactive or all-pass state; i.e. QMF302 is operable to pass ions having any m/z value therethrough. Finally,a spectrum 456 of ion drift time (corresponding to ion mobility) vs. ionflight time (corresponding to ion mass) is shown in FIG. 14Dillustrating one example of the resulting ion spectrum of step 412.

Close inspection of spectrum 456 of FIG. 14D reveals that ions a, b andg do not overlap in drift times with any other ion, while ions c and dand ions e and f overlap in their respective drift times. Ions c and dwill accordingly arrive at collision cell 304 at approximately the sametime (3.5 μs), and ions e and f will accordingly arrive at collisioncell 304 at approximately the same time (4.8 μs). If collision cell 304was filled with buffer gas so that ion fragmentation occurred, TOFMS 36would not be able to accurately distinguish parent and daughter ionsattributable to ion c from those of ion d and likewise thoseattributable to ion e from those of ion f. If, however, no such overlapsoccurred, the foregoing problem would not occur. In accordance with animportant aspect of the present invention, process 400 is configured toconduct subsequent sequencing analysis (via fragmentation) with QMF 302operating in an all-pass mode if no overlap in ion drift times areevident from step 412, but is alternatively operable to conductsubsequent sequencing analysis (via fragmentation) with QMF 302 operableto selectively filter out all but one of the ions overlapping in any onedrift time. In the latter case, the sequencing analysis is repeateduntil fragmentation spectra are produced for all ions in the originalspectrum (FIG. 14D). Thus in the example of FIG. 14D, sequencinganalysis is conducted by filling collision cell 304 with buffer gas andoperating QMF 302 to selectively filter out ions d and f, for example,such that the resulting fragmentation spectrum includes fragmentationspectra of ions a, b, c, e and g. The sequencing analysis is repeated bycontrolling QMF 302 to selectively filter out ions c and e such that theresulting fragmentation spectrum includes fragmentation spectra of atleast ions d and f. In general, the instrument 300 must be taken throughan ion generation/resulting spectrum sequence Z+1 times for any sample,wherein Z is the maximum number of ions overlapping in drift time andthe “1” accounts for the initial operation of instrument 300 in order toproduce the spectrum 456 of FIG. 14D. In the example illustrated inFIGS. 14, 15 and 16, instrument 300 must accordingly be taken throughthe ion generation/resulting spectrum sequence three times since themaximum number of ions overlapping in drift time is two (e.g., two ionsc and d overlap in drift time and, two ions f and e overlap in drifttime).

Referring again to FIG. 13, process 400 continues from step 412 and step414 where process 400 is directed to the subprocess flagged with thecurrent value of A. In the first time through process 400, A=1 soprocess 400 jumps to step 416. Thereafter at step 418, the collisioncell 304 is filled with buffer gas from buffer gas source 306 (or buffergas source 46). As with step 404, step 418 may be executed manually orunder the control of computer 310. In either case, process 420 advancesfrom step 418 to step 420 where a determination is made as to whetherthere exists any overlap in ion packet drift times. Step 420 ispreferably carried out by manually observing spectrum 456 (FIG. 14D),although the present invention contemplates that step 420 may beautomated in accordance with known techniques and therefore executed bycomputer 310. In either case, if no overlap in ion drift times arepresent in the spectrum resulting at step 412, steps 408-412 arerepeated and a spectrum of fragmented parent and daughter ions results,wherein the spectrum of fragmented parent and daughter ions may beanalyzed further for sequencing purposes. If, however, ion drift timeoverlap is observed in the first execution of step 412, process 400continues from step 420 at step 422 where QMF 302 is configured toselectively filter out desired m/z values based on the observedoverlapping drift times. Thereafter, the process counter A isincremented and steps 408-412 are repeated.

Referring now to FIGS. 15A-15D, step 422 and a second pass through steps408, 410 and 412 are illustrated. The ion gate signal 450 and TOFMSsignals 452 are identical to those shown in FIGS. 14A and 14B, but theQMF signal 458 includes an activation pulse 458 ₁ during a time periodencompassing the drift times of ions c and d, and an activation pulse458 ₂ encompassing the drift times of ions e and f. It is to beunderstood that activation pulses 458 ₁ and 458 ₂ are not meant torepresent a single-signal activation of QMF 302 (i.e., “triggering”),but are instead meant to represent the activation times of QMS 302relative to known ion drift times, wherein computer 302 is operableduring each of these activation times to control the voltage sources 320₁ and 320 ₂ (FIG. 11), as described hereinabove, to thereby pass onlyions having a desired m/z value and to filter out ions having any otherm/z value. In the example spectrum illustrated in FIG. 15D, computer 310is operable to control QMF 302 during activation time 458 ₁ to pass onlyions having m/z values equal to that of ion c so that ion d iseffectively filtered out. Similarly, computer 310 is operable to controlQMF 302 during activation time 458 ₂ to pass only ions having m/z valuesequal to that of ion e so that ion f is effectively filtered out. In onepreferred embodiment of process 400, computer 310 is operable at allother times in an all-pass mode to thereby pass therethrough ions havingany m/z value. In an alternate embodiment, computer 310 may be operableto sequentially control QMF 302 during time periods corresponding to thedrift times of each of the ions, wherein computer 310 is operable duringsuch time periods to pass only ions having m/z values equal to those ofinterest. Thus, for the example spectrum 460 illustrated FIG. 15D,computer 310 may alternatively be operable to activate QMF 302 duringthe drift time of ion a to pass only ions having m/z values equal tothat of ion a, to activate QMF 302 during the drift time of ion b tothereby pass only ions having m/z values equal to that of ion b, toactivate QMF 302 during the drift time of ions c and d to pass only ionshaving m/z values equal to that of ion c, etc. In either case, thespectrum 460 of FIG. 15D results, wherein the flight times of each ofthe parent and daughter ions resulting from the fragmentation of ions a,b, c, e and g in collision cell 304 are clearly resolved. From theseflight times, the m/z values of each of the fragmented ions may bedetermined in accordance with known techniques.

Referring again to FIG. 13, process 400 advances from a second executionof step 412 to step 414 where process 400 is directed to a processsection flagged by the most recent value of the counting variable A. Inthis case, A=2 so process 400 is directed to step 426. Thereafter atstep 428, a determination is made as to whether any ion packets existthat have not yet been accounted for in the spectrum 460 of FIG. 15D. Inone preferred embodiment, step 428 is conducted manually via examinationof spectra 456 and 460, although the present invention contemplates thatstep 428 may alternatively be automated in a known manner andaccordingly be executed by computer 310. In any case, if it isdetermined at step 428 that no ion packets are unaccounted for, process400 advances to step 432 where process 400 is terminated. If, on theother hand, it is determined at step 428 that there exists at least oneion packet that has not yet been accounted for in spectrum 460, process400 advances to step 430 where QMF 302 is configured to selectivelyfilter out desired m/z values based on the observed overlapping drifttimes. Thereafter, steps 408-412 are again repeated.

Referring now to FIGS. 16A-16D, step 430 and a third pass through steps408, 410 and 412 are illustrated. The ion gate signal 450 and TOFMSsignals 452 are identical to those shown in FIGS. 14A and 14B, but theQMF signal 462 includes an activation pulse 462 ₁ during a time periodencompassing the drift times of ions c and d, and an activation pulse462 ₂ encompassing the drift times of ions e and f. Again, it is to beunderstood that activation pulses 462 ₁ and 462 ₂ are not meant torepresent a single-signal activation of QMF 302 (i.e., “triggering”),but are instead meant to represent the activation times of QMS 302relative to known ion drift times, wherein computer 302 is operableduring each of these activation times to control the voltage sources 320₁ and 320 ₂ (FIG. 11), as described hereinabove, to thereby pass onlyions having a desired m/z value and to filter out ions having any otherm/z value. In the example spectrum illustrated in FIG. 16D, computer 310is operable to control QMF 302 during activation time 462 ₁ to pass onlyions having m/z values equal to that of ion d so that ion c iseffectively filtered out. Similarly, computer 310 is operable to controlQMF 302 during activation time 462 ₂ to pass only ions having m/z valuesequal to that of ion f so that ion e is effectively filtered out. In onepreferred embodiment of process 400, computer 310 is operable at allother times in a no-pass mode to thereby inhibit passage therethrough ofions having any m/z value. In an alternate embodiment, computer 310 maybe operable to sequentially control QMF 302 during time periodscorresponding to the drift times of each of the ions, wherein computer310 is operable during such time periods to pass only ions having m/zvalues equal to those of interest. Thus, for the example spectrum 464illustrated FIG. 16D, computer 310 may additionally be operable toactivate QMF 302 during the drift times of ions a, b and g to pass onlyions having m/z values equal to those of ions a, b and g respectively.This will result in redundant flight time information forparent/daughter ions of a, b and g, but such operation serves as anaccuracy check on the data obtained from spectrum 464. In the firstcase, the spectrum 464 of FIG. 16D results, wherein the flight times ofeach of the parent and daughter ions resulting from the fragmentation ofions d and f in collision cell 304 are clearly resolved. In the lattercase, a spectrum similar to spectrum 460 of FIG. 15D results, whereinthe flight times of each of the parent and daughter ions resulting fromthe fragmentation of ions a, b, d, f and g in collision cell 304 areclearly resolved. In either case, the m/z values of each of thefragmented ions may be determined from their associated flight times inaccordance with known techniques.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly the preferred embodiments have been shown and described and thatall changes and modifications that come within the spirit of theinvention are desired to be protected. For example, referring to FIG.17, alternative variations of the ion mobility and mass spectrometerinstrument of FIG. 9 are illustrated, wherein ion trapping, ion massfiltering and ion fragmentation functions may, in accordance with thepresent invention, be positioned in various locations with respect tothe ion source 32, ion mobility instrument 34 and time-of-flight massspectrometer 36. In a first specific example, structure 500 represents aquadrupole mass filter, such as QMF 302 described hereinabove,structures 502 and 504 may be omitted, and structure 506 represents acollision cell such as collision cell 304. In this embodiment, ion massselection is performed prior to injecting ions into IMS 34, and ionfragmentation is performed between IMS 34 and TOFMS 36. In a secondspecific example, structure 500 represents a quadrupole mass filter,such as QMF 302 described hereinabove, structure 502 represents an iontrap, such as ion trap 152 described hereinabove, structure 504 isomitted and structure 506 represents a collision cell such as collisioncell 304 described hereinabove. In this embodiment, mass selection isperformed upon ions generated by ion source 32 and the mass selectedions are collected in the ion trap 152 prior to injection into IMS 34.Fragmentation is performed in collision cell 304 as describedhereinabove. Additionally, or alternatively, fragmentation may also beperformed in ion trap 152, as is known in the art, if ion trap 152 issupplied with a suitable buffer gas (not shown) and/or in IMS 34 asdescribed hereinabove. In a third specific example, structure 500represents a quadrupole mass filter, such as QMF 302 describedhereinabove, structure 502 represents a collision cell such as collisioncell 304 described hereinabove, and structures 504 and 506 are omitted.In this embodiment, mass selection is performed upon ions generated byion source 32 and the mass selected ions are fragmented in collisioncell 304 prior to injection into IMS 34. Fragmentation may additionallyor alternatively be performed in IMS 34, and/or an additional collisioncell 304 may be provided as structure 506 for further fragmenting theions supplied by IMS 34. In a fourth specific example, structure 500represents a quadrupole mass filter, such as QMF 302 describedhereinabove, structure 502 represents an ion trap, such as ion trap 152described hereinabove, structure 504 represents a collision cell, suchas collision cell 304 described hereinabove, and structure 506 isomitted. In this embodiment, mass selection is performed upon ionsgenerated by ion source 32, followed by collection of the mass filteredions within ion trap 152, followed by fragmentation of the ionscollected in trap 152 either within trap 152 and/or within collisioncell 304 prior to injection of the ions into IMS 34. Furtherfragmentation may be performed within IMS 34 and/or structure 506 maydefine an additional collision cell for further ion fragmentation priorto injection of the ions into TOFMS 36. Generally, it is to beunderstood that ion mass selection and ion fragmentation may occur atvarious and multiple locations relative to ion source 32, IMS 34 andTOFMS 36. Moreover, it is to be understood that IMS 34 may be generallyconfigured as a known gas chromatograph, as illustrated hereinabove, oralternatively as a known liquid chromatograph, without detracting fromthe scope of the present invention.

What is claimed is:
 1. A method of generating ion mass spectralinformation, comprising the steps of: generating gaseous ions from asample source; collecting at least some of said generated gaseous ionsin an ion trap; repeating said generating and collecting steps a numberof times to thereby form a gaseous bulk of ions in the ion trap;releasing said gaseous bulk of ions from said ion trap; gating at leasta portion of said bulk of ions into an ion mobility spectrometer tothereby separate said bulk of ions in time to form a number of ionpackets each having an ion mobility associated therewith; sequentiallydirecting at least some of said ion packets into a mass spectrometer;continually activating said mass spectrometer to thereby sequentiallyseparate at least some of said ion packets in time to form a number ofion subpackets each having an ion mass associated therewith; andprocessing at least some of the ion subpackets to determine massspectral information therefrom.
 2. The method of claim 1 wherein thestep of generating gaseous ions from a sample source includes generatingsaid gaseous ions via electrospray ionization.
 3. The method of claim 1wherein the step of generating gaseous ions from a sample sourceincludes generating said gaseous ions via laser desorption ionization.4. A method of generating ion mass spectral information, comprising thesteps of: generating a gaseous bulk of ions; gating at least a portionof said bulk of ions into an ion mobility spectrometer to therebyseparate said bulk of ions in time to form a number of ion packets eachhaving an ion mobility associated therewith; sequentially directing atleast some of said ion packets into a mass spectrometer; continuallyactivating said mass spectrometer to thereby sequentially separate atleast some of said ion packets in time to form a number of ionsubpackets each having an ion mass associated therewith; processing atleast some of the ion subpackets to determine mass spectral informationtherefrom; and sequentially fragmenting said ion packets into daughterions prior to sequentially directing at least some of said ion packetsinto a mass spectrometer.
 5. A method of generating ion mass spectralinformation, comprising the steps of: generating a gaseous bulk of ions;gating at least a portion of said bulk of ions into an ion mobilityspectrometer to thereby separate said bulk of ions in time to form anumber of ion packets each having an ion mobility associated therewith;sequentially directing at least some of said ion packets into a massspectrometer; continually activating said mass spectrometer to therebysequentially separate at least some of said ion packets in time to forma number of ion subpackets each having an ion mass associated therewith;processing at least some of the ion subpackets to determine massspectral information therefrom; and selectively filtering said ionpackets to thereby sequentially provide ion packets having only desiredmass-to-charge ratios prior to sequentially directing at least some ofsaid ion packets into a mass spectrometer.
 6. The method of claim 5further including the step of sequentially fragmenting said ion packetshaving only only desired mass-to-charge ratios into daughter ions priorto sequentially directing at least some of said ion packets into a massspectrometer.
 7. A method of generating ion mass spectral information,comprising the steps of: generating a gaseous bulk of ions; gating atleast a portion of said bulk of ions into an ion mobility spectrometerto thereby separate said bulk of ions in time to form a number of ionpackets each having an ion mobility associated therewith; sequentiallydirecting at least some of said ion packets into a mass spectrometer;continually activating said mass spectrometer to thereby sequentiallyseparate at least some of said ion packets in time to form a number ofion subpackets each having an ion mass associated therewith; processingat least some of the ion subpackets to determine mass spectralinformation therefrom; and fragmenting said gaseous bulk of ions intodaughter ions prior to said gating step.
 8. A method of generating ionmass spectral information, comprising the steps of: generating a gaseousbulk of ions; gating at least a portion of said bulk of ions into an ionmobility spectrometer to thereby separate said bulk of ions in time toform a number of ion packets each having an ion mobility associatedtherewith; sequentially directing at least some of said ion packets intoa mass spectrometer; continually activating said mass spectrometer tothereby sequentially separate at least some of said ion packets in timeto form a number of ion subpackets each having an ion mass associatedtherewith; processing at least some of the ion subpackets to determinemass spectral information therefrom; and selectively filtering saidgaseous bulk of ions to thereby provide a gaseous bulk of ions havingonly desired mass-to-charge ratios prior to said gating step.
 9. Themethod of claim 8 further including the step of fragmenting said gaseousbulk of ions having only desired mass-to-charge rations into daughterions prior to said gating step.
 10. The method of claim 9 furtherincluding the step of sequentially fragmenting said ion packets intodaughter ions prior to sequentially directing at least some of said ionpackets into a mass spectrometer.
 11. Apparatus for generating massspectral information from a sample source, comprising: means forgenerating a gaseous bulk of ions from a sample source; an ion mobilityspectrometer (IMS) having an ion inlet coupled to said means forgenerating a gaseous bulk of ions and an ion outlet, said IMS operableto separate ions in time as a function of ion mobility; a massspectrometer (MS) having an ion acceleration region coupled to said ionoutlet of said IMS and an ion detector, said MS operable to separateions in time as a function of ion mass; a collision cell disposedbetween said ion outlet of said IMS and said acceleration region of saidMS, said collision cell having a buffer gas therein operable to fragmentparent ions provided at said outlet of said IMS into daughter ions priorto entrance into said ion acceleration region of said MS; and a computeroperable to gate at least a portion of said gaseous bulk of ions intosaid ion inlet of said IMS and to continually pulse said ionacceleration region of said MS to thereby sequentially direct ionstoward said ion detector.
 12. Apparatus for generating mass spectralinformation from a sample source, comprising: means for generating agaseous bulk of ions from a sample source; an ion mobility spectrometer(IMS) having an ion inlet coupled to said means for generating a gaseousbulk of ions and an ion outlet, said IMS operable to separate ions intime as a function of ion mobility; a mass spectrometer (MS) having anion acceleration region coupled to said ion outlet of said IMS and anion detector, said MS operable to separate ions in time as a function ofion mass; an ion mass filter disposed between said ion outlet of said MSand said ion acceleration region of said MS, said ion mass filterdirecting ions having only desired mass-to-charge ratios into said ionacceleration region of said MS; and a computer operable to gate at leasta portion of said gaseous bulk of ions into said ion inlet of said IMSand to continually pulse said ion acceleration region of said MS tothereby sequentially direct ions toward said ion detector.
 13. Theapparatus of claim 12 further including a collision cell disposedbetween said ion mass filter and said ion acceleration region of saidMS, said collision cell having a buffer gas therein operable to fragmentparent ions having only desired mass-to-charge ratios into daughter ionsprior to entrance into said ion acceleration region of said MS. 14.Apparatus for generating mass spectral information from a sample source,comprising: means for generating a gaseous bulk of ions from a samplesource; an ion mobility spectrometer (IMS) having an ion inlet coupledto said means for generating a gaseous bulk of ions and an ion outlet,said IMS operable to separate ions in time as a function of ionmobility; a collision cell disposed between said means for generating agaseous bulk of ions and said ion inlet of said IMS, said collision cellhaving a buffer gas therein operable to fragment parent ions provided bysaid means for generating a gaseous bulk of ions into daughter ionsprior to entrance into said ion inlet of said IMS; a mass spectrometer(MS) having an ion acceleration region coupled to said ion outlet ofsaid IMS and an ion detector, said MS operable to separate ions in timeas a function of ion mass; and a computer operable to gate at least aportion of said gaseous bulk of ions into said ion inlet of said IMS andto continually pulse said ion acceleration region of said MS to therebysequentially direct ions toward said ion detector.
 15. Apparatus forgenerating mass spectral information from a sample source, comprising:means for generating a gaseous bulk of ions from a sample source; an ionmobility spectrometer (IMS) having an ion inlet coupled to said meansfor generating a gaseous bulk of ions and an ion outlet, said IMSoperable to separate ions in time as a function of ion mobility; an ionmass filter disposed between said means for generating a gaseous bulk ofions and said ion inlet of said IMS, said ion mass filter directing ionshaving only desired mass-to-charge ratios into said ion inlet of saidIMS; a mass spectrometer (MS) having an ion acceleration region coupledto said ion outlet of said IMS and an ion detector, said MS operable toseparate ions in time as a function of ion mass; and a computer operableto gate at least a portion of said gaseous bulk of ions into said ioninlet of said IMS and to continually pulse said ion acceleration regionof said MS to thereby sequentially direct ions toward said ion detector.16. The apparatus of claim 15 further including a collision celldisposed between said ion mass filter and said ion inlet of said IMS,said collision cell having a buffer gas therein operable to fragmentparent ions provided by said ion mass filter into daughter ions prior toentrance into said ion inlet of said IMS.
 17. A method of generating ionmass spectral information, comprising the steps of: generating a gaseousbulk of ions; separating said gaseous bulk of ions in time as a functionof ion mobility; where two or more ions overlap in ion mobility values,filtering out ions that have all but a desired mass-to-charge ratio;sequentially separating in time the post-filtered ions as a function ofion mass; and processing ions separated in time as functions of ionmobility and ion mass to determine ion mass spectral informationtherefrom.
 18. The method of claim 17 further including the step offragmenting post-filtered parent ions into daughter ions prior to thestep of sequentially separating the post filtered ions as a function ofion mass.
 19. The method of claim 18 wherein the step of generating agaseous bulk of ions includes generating said gaseous bulk of ions viaelectrospray ionization.
 20. The method of claim 18 wherein the step ofgenerating a gaseous bulk of ions includes generating said gaseous bulkof ions via laser desorption ionization.
 21. The method of claim 18wherein the step of generating a gaseous bulk of ions includes the stepsof: generating gaseous ions from a sample source; collecting at leastsome of said generated gaseous ions in an ion trap; repeating saidgenerating and collecting steps a number of times to thereby form agaseous bulk of ions in the ion trap; and releasing said gaseous bulk ofions from said ion trap.
 22. The method of claim 18 wherein the step ofgenerating a gaseous bulk of ions includes the steps of: continuallygenerating gaseous ions from a sample source; and collecting a bulk ofsaid continually generated gaseous ions in an ion collection chamber;and releasing at least a portion of said continually generated gaseousions from said ion collection chamber.
 23. The method of claim 22wherein the step of generating gaseous ions from a sample sourceincludes generating said gaseous ions via electrospray ionization. 24.The method of claim 22 wherein the step of generating gaseous ions froma sample source includes generating said gaseous ions via laserdesorption ionization.
 25. Apparatus for generating mass spectralinformation from a sample source, comprising: means for generating agaseous bulk of ions from a sample source; an ion mobility spectrometer(IMS) having an ion inlet coupled to said means for generating a gaseousbulk of ions and an ion outlet, said IMS operable to separate ions intime as a function of ion mobility; an ion filter having a filter inletcoupled to said ion outlet of said IMS and a filter outlet, said ionfilter operable to sequentially pass therethrough only ions havingdesired mass-to-charge ratios; and a mass spectrometer (MS) having anion acceleration region coupled to said filter outlet and an iondetector, said MS operable to sequentially separate in time ionsprovided thereto by said ion filter as a function of ion mass.
 26. Theapparatus of claim 25 further including a computer operable to gate atleast a portion of said gaseous bulk of ions into said ion inlet of saidIMS and to continually pulse said ion acceleration region of said MS.27. The apparatus of claim 26 wherein said computer is further operableto control said filter to thereby permit passage therethrough of ionshaving only desired mass-to-charge ratios.
 28. The apparatus of claim 27wherein said means for generating a bulk of gaseous ions is responsiveto a gate signal to generate said gaseous bulk of ions; and wherein saidcomputer is operable to produce said gate signal to thereby gate atleast a portion of said gaseous bulk of ions into said ion inlet of saidIMS.
 29. The apparatus of claim 28 wherein said ion inlet of said IMSdefines an ion gate responsive to an active gate signal to permitentrance of gaseous ions into said ion inlet of said IMS and tootherwise inhibit entrance of gaseous ions into said ion inlet of saidIMS; and wherein said computer is operable to control said gate signal,said control computer activating said gate signal for a programmabletime period to thereby gate at least a portion of said gaseous bulk ofions into said ion inlet of said IMS.
 30. The apparatus of claim 25further including a collision cell disposed between said ion filteroutlet and said acceleration region of said MS, said collision cellhaving a buffer gas therein operable to fragment ions provided at saidion filter outlet into parent and daughter ions prior to entrance intosaid ion acceleration region of said MS.
 31. A method of generating ionmass spectral information, comprising the steps of: generating a gaseousbulk of ions; separating said gaseous bulk of ions in time as a functionof ion mobility; sequentially separating in time as a function of ionmass each of the ions separated in time as a function of ion mobility;processing ions separated in time as functions of ion mobility and ionmass to determine ion mass spectral information therefrom; repeating thegenerating and separating steps followed by the step of sequentiallyfragmenting into daughter ions each of the ions separated in time as afunction of ion mobility, followed by the sequentially separating andprocessing steps only if the initial processing step indicates that notwo or more ions overlap in mobility values.
 32. The method of claim 31further including the step of filtering out ions that have all but adesired mass-to-charge ratio, followed by repeating the generating andseparating steps, followed by the step of sequentially fragmenting intodaughter ions each of the ions separated in time as a function of ionmobility, followed by the sequentially separating and processing stepsonly if the previous processing step indicates that two or more ionsoverlap in mobility values.
 33. The method of claim 32 further includingthe step of repeating the filtering step until all ions overlapping inion mobility values have been processed.
 34. The method of claim 33wherein the step of generating a gaseous bulk of ions includesgenerating said gaseous bulk of ions via electrospray ionization. 35.The method of claim 33 wherein the step of generating a gaseous bulk ofions includes generating said gaseous bulk of ions via laser desorptionionization.
 36. The method of claim 33 wherein the step of generating agaseous bulk of ions includes the steps of: generating gaseous ions froma sample source; collecting at least some of said generated gaseous ionsin an ion trap; repeating said generating and collecting steps a numberof times to thereby form a gaseous bulk of ions in the ion trap; andreleasing said gaseous bulk of ions from said ion trap.
 37. The methodof claim 33 wherein the step of generating a gaseous bulk of ionsincludes the steps of: continually generating gaseous ions from a samplesource; and collecting a bulk of said continually generated gaseous ionsin an ion collection chamber; and releasing at least a portion of saidcontinually generated gaseous ions from said ion collection chamber. 38.The method of claim 37 wherein the step of generating gaseous ions froma sample source includes generating said gaseous ions via electrosprayionization.
 39. The method of claim 37 wherein the step of generatinggaseous ions from a sample source includes generating said gaseous ionslaser desorption ionization.